DUSP1 and SOX2 expression determine squamous cell carcinoma of the salivary gland progression

Salivary gland squamous cell carcinomas (SG-SCCs) constitute a rare type of head and neck cancer which is linked to poor prognosis. Due to their low frequency, the molecular mechanisms responsible for their aggressiveness are poorly understood. In this work we studied the role of the phosphatase DUSP1, a negative regulator of MAPK activity, in controlling SG-SCC progression. We generated DUSP1 KO clones in A253 human cells. These clones showed a reduced ability to grow in 2D, self-renew in ECM matrices and to form tumors in immunodeficient mice. This was caused by an overactivation of the stress and apoptosis kinase JNK1/2 in DUSP1−/+ clones. Interestingly, RNAseq analysis revealed that the expression of SOX2, a well-known self-renewal gene was decreased at the mRNA and protein levels in DUSP1−/+ cells. Unexpectedly, CRISPR-KO of SOX2 did not recapitulate DUSP1−/+ phenotype, and SOX2-null cells had an enhanced ability to self-renew and to form tumors in mice. Gene expression analysis demonstrated that SOX2-null cells have a decreased squamous differentiation profile -losing TP63 expression- and an increased migratory phenotype, with an enhanced epithelial to mesenchymal transition signature. In summary, our data indicates that DUSP1 and SOX2 have opposite functions in SG-SCC, being DUSP1 necessary for tumor growth and SOX2 dispensable showing a tumor suppressor function. Our data suggest that the combined expression of SOX2 and DUSP1 could be a useful biomarker to predict progression in patients with SG-SCCs.

www.nature.com/scientificreports/presented as disorganized with very few squamous structures in comparison with control tumors (Fig. 2B).DUSP1 −/+ tumors showed an increased number of Caspase 3-positive cells (Fig. 2C, D), but surprisingly, DUSP1 −/+ tumors still showed no difference in the expression of proliferative markers KI67 and pH3 (Figure Supp 2A, B).To better understand this phenotype, we subjected cells from control and DUSP1 −/+ clones to RNAseq.We performed differential gene expression (DGE) analysis comparing control and DUSP1 −/+ clones and chose only the genes that concordantly changed in both 1 and 10 DUSP1 -/+ clones (Figure Supp 2C, Table Supp.1).This analysis revealed 269 genes upregulated and 248 genes downregulated in both DUSP1 −/+ clones (Fig. 2E).Increased genes represented mainly processes related to keratinization (Cornification, epidermal cell differentiation) with genes such as FGL, IVL, HRNR, KRT1 or KRT4 (Fig. 2F, G) being increased in the absence of DUSP1.On the other hand, downregulated genes included pathways related with cell migration such as focaladhesion kinase and PI3K-AKT, and biological processes such as locomotion (Fig. 2F).Genes of these pathways included well-known regulators of epithelial to mesenchymal transition (EMT) such as ZEB1 and TWIST1 and their regulated genes FN1 and VIM (Fig. 2G).We validated the increased expression of keratinization related genes by qPCR, demonstrating an increased expression of KRT1, IVL, FLG or KRT4 (Figure Supp 2D).Additionally, we measured KRT10 protein levels detecting higher expression of KRT10 in DUSP1 -/+ tumors in comparison to the control (Fig. 2H).Interestingly, the areas occupied by KRT10 positive cells were smaller in DUSP1 −/+ cells, confirming our original observation of decreased keratin pearls.This may suggest that in the absence of DUSP1, cells differentiate expressing higher levels of KRT, but fail to expand and form keratin pearls.Vimentin (VIM) staining in organoid sections, as well as in tumor sections demonstrated a marked reduction in vimentin expression, specifically in cancer cells (Fig. 2I, J) from both DUSP1 −/+ clones, validating the previous results.Altogether, these data show that DUSP1 deficiency promotes a more differentiated, squamous phenotype.

SOX2 does not mediate DUSP1 phenotype
Since DUSP1 KO clones had reduced self-renewal capacity in vitro and in vivo, we looked for known self-renewal transcription factors downregulated in both DUSP1 -/+ clones, identifying SOX2 (Fig. 3A, Figure Supp 2D).We validated that SOX2 protein was downregulated to almost undetectable protein levels in all DUSP1 −/+ clones that we generated (Fig. 3B), showing that this effect was independent of the clone.This was confirmed using BCI treatment, which also decreased SOX2 protein levels (Figure Supp.2E).Finally, we observed a strong decrease in SOX2 protein level in DUSP1 −/+ tumors in comparison with the control (Fig. 3C).These data suggest that DUSP1 −/+ tumor cells are more differentiated, and this could be due to a loss of the self-renewal transcription factor (TF) SOX2 as it was described in the cutaneous SCC (cSCC) context 14 .
To distinguish if the decreased SOX2 expression could be driving DUSP1 −/+ phenotype, we rescued SOX2 expression on DUSP1 −/+ Cl10 using a lentiviral construct.After we confirmed the correct increase in SOX2  www.nature.com/scientificreports/protein on DUSP1 −/+ Cl10 (Fig. 3D), we performed organoid cultures and measured their growth at the endpoint of the experiment.While control organoids grew significantly bigger with SOX2 OE, DUSP1 −/+ Cl10 organoids grew even smaller upon SOX2 OE (Fig. 3E).Western blot analysis of organoid protein lysate demonstrated that DUSP1 −/+ Cl10 organoids express higher levels of pJNK than controls, and this could not be rescued by SOX2 OE (Fig. 3F).We concluded that increased JNK activation rather than SOX2 loss of expression was responsible of the cell death induced after DUSP1 loss.

SOX2 silencing enhances SG-SCC cell proliferation and tumor growth
To further test the importance of SOX2 in SG-SCC development, we mutated SOX2 gene using CRISPR/Cas9 and two independent sgRNAs.sgRNA2 produced a complete loss of SOX2 protein, while with sgRNA1 maintained some SOX2 protein (Fig. 4A).Since SOX2 has been described to control cell proliferation, we first performed cell growth assays.Accordingly with its known role, we detected a reduction in cell growth upon SOX2 silencing (Fig. 4B).Next, we measured the effects of SOX2 loss in self-renewal by performing organoid formation assays in ECM matrixes.Surprisingly, we observed a significant increase in the size of organoid formed by sgSOX2 cells (Fig. 4C, D).This increase was dependent on the levels of SOX2 protein since it was more significant in sgSOX2.2than in sgSOX2.1 cells.We also observed a significant increase in the number of organoids in sgSOX2.1 cells.H&E staining of these organoids suggested that sgSOX2 organoids contained less differentiated-like structures than sgTomato cells (Fig. 4C) but had similar cell density (Figure Supp.3A).In addition, sgSOX2.2cells contained higher number of KI67-positive cells (Fig. 4E, E').Unexpectedly, these data suggest that SOX2 loss enhances cancer cell self-renewal capacity in SG-SCCs.
To determine if this was also true in vivo, we injected sgTomato and sgSOX2 cells intradermically into Nu/Nu mice to measure tumor growth.Both sgSOX2-derived tumors grew faster than control sgTomato tumors (Fig. 4F).Tumor growth was dependent on SOX2 levels, since sgSOX2.1 tumors that still expressed some levels of SOX2 protein grew only slightly faster than the controls, while sgSOX2.2tumors (Fig. 4G), which have no detectable SOX2 protein expression, grew much faster than controls.Consistently, we quantified higher pH3 and KI67 levels in sgSOX2.2tumors (Fig. 4H, Figure Supp.3B), and only a tendency in sgSOX2.1 tumors in comparison to the sgTomato controls.Overall, these data demonstrate that SOX2 loss promotes tumor growth in A253-derived SG-SCCs.These results contrast with those obtained for DUSP1 mutant cells that also presented low SOX2 levels, reinforcing that the DUSP1 phenotype is not mediated by SOX2 loss.
To further understand the interconnection between DUSP1 and SOX2, we measured DUSP1 expression on SOX2 deficient cells upon U.V. irradiation (Figure Supp 3C).Interestingly, DUSP1 levels are decreased both in basal conditions and upon U.V. irradiation in sgSOX2 cells.Reanalyzing SOX2 ChIPseq data in HNSCCs 16 , we identified that DUSP1 is a SOX2 direct target (Figure Supp 3D), which can explain why it is downregulated in sgSOX2 cells.
Intriguingly, when we cause DUSP1 loss as the first event, SOX2 is downregulated and there is an overactivation of JNK signaling in comparison to control cells in response to U.V. or growth in 3D conditions (Figure Supp 3C, Fig. 3F).In contrast, when SOX2 expression is lost as the first event, DUSP1 expression is decreased but pJNK levels remain similar to sgTomato control cells (Figure Supp 3C).This data suggests that when SOX2 is lost, DUSP1 expression may be dispensable since JNK signaling is downregulated, and in the absence of pJNK activation, cells can avoid apoptosis.

SOX2 loss transcriptionally reprograms SG-SCCs to a more mesenchymal cell phenotype
To further understand the phenotype produced by SOX2 loss, we performed RNAseq comparing control to sgSOX2.2cells.We rationalized that to exert a full effect we needed to have a situation with no SOX2 protein, since low levels of transcription factors (TFs) can still efficiently control gene expression.We therefore used sgSOX2.2cells for this analysis.SOX2 loss produced large changes in gene expression (Fig. 5A, Table Supp.2), with 735 up-regulated and 279 down-regulated genes.Among the downregulated pathways and processes, there was a decrease in cytodifferentiation (Fig. 5B), with a decreased expression of multiple keratin genes, such as KRT14, KRT5 or KRT1 among many others (Fig. 5C).qPCR validation, confirmed these changes (Figure Supp.3E), and staining for KRT10, identified lower positive areas with decreased expression of KRT10 (Fig. 5D).Three processes and pathways were particularly enriched in SOX2 null cells: cell migration, vascularization, and response to cytokines (Fig. 5B).Genes of the first two pathways included TGFbeta pathway genes TGFB2, EMT genes such as VIM, FN1, SERPINE2, ZEB1 or LOXL2, and VEGFA, VEGFC or PDGFB respectively (Fig. 5E) Figure Supp.3D).We could confirm an increase of vimentin in sgSOX2 derived organoids (Fig. 5F), and sgSOX2 cells became more migratory in 2D scratch assays (Fig. 5G).We also observed a pronounced decrease in pathways related to protein translation and the endoplasmic reticulum, and particularly in OXPHOS, suggesting that SOX2 null cells are suffering a metabolic reprograming that could be guiding the aggressiveness of these cells.Remarkably, when we compared the DEG of DUSP1 or SOX2 loss of function, we find out that they control completely opposite functions (Figure Supp 3F).Keratinization genes are increased in DUSP1 -/+ cells and decreased in sgSOX2 cells, while EMT and migration genes are decreased in DUSP1 -/+ cells and increased in sgSOX2 cells.These data reinforce the idea that SOX2 and DUSP1 control opposite functions during tumors progression in SG-SCCs.
Since we and others had shown that SOX2 controls TP63 expression 16 , and TP63 defines the expression of squamous programs in cSCCs, we analyzed the expression of TP63 in sgSOX2 tumors.We observed a SOX2dose dependent decrease in TP63 expression, suggesting that squamous features may be lost by a concomitant decrease in TP63 and SOX2 expression (Fig. 5H).The decrease in TP63 protein was dependent on growth conditions, since 2D cultures showed unaltered protein levels, which became undetectable when we grew sgTomato or sgSOX2 cells in 3D, that better resemble tumor conditions (Figure Supp 3G).Intrigued by this observation, we explored the expression of other stem related TFs, and we identified that the expression of BMI1 17 , KLF4 18 and HMGA2 was significantly increased in sgSOX2 cells (Fig. 5I).Overall, these data suggest that SOX2 null SG-SCC are reprogramed to a more mesenchymal phenotype and migratory capacity, with loss of TP63 and squamous markers, which could explain its more aggressive behavior.

Discussion
Salivary gland squamous cell carcinomas are a very rare type of cancer that can originate from the basal cells of the salivary gland, having a bad prognosis in comparison to other salivary gland malignancies 8 .In this work we aimed to investigate the function of a pleiotropic protein, the protein phosphatase DUSP1, which we have shown, among others, to have pro-oncogenic or tumor suppressor function depending on the cancer type 13 .We generated DUSP1 CRISPR-KO A253 cells, which were described as well-differentiated SG-SCCs.We found that DUSP1 −/+ cells had a predominant stress phenotype, caused probably by an increased JNK activity, which made these cells unable to grow 3D in ECM matrix.We observed that many of these cells suffered apoptosis in 3D, and when transplanted into mice, formed very small and disorganized tumors.Indeed, we were only able to generate heterozygous deletions of DUSP1 gene, highlighting its essential role in this cancer type.Although heterozygous, some of the clones did not express DUSP1 protein, which could be due to the introduction of inactivating indels in WT alleles that prevented the correct DUSP1 expression.This agrees with the phenotype that we observed in lung 19,20 and that was observed in other cancer types, where DUSP1 expression increases early during tumorigenesis and prevents JNK-mediated apoptosis.Importantly, we could recapitulate some of these findings using a DUSP1 inhibitor, highlighting the translational potential of our results.
The RNAseq analysis of DUSP1 −/+ cells revealed that these cells have an increased expression of genes related to epithelial keratinization, and therefore a more differentiated phenotype.This phenotype has been shown to be linked to the loss of self-renewal capacity in cutaneous SCCs (cSCCs), through the loss of expression of the transcription factor PITX1 16 .Indeed, DUSP1 −/+ cells lose the expression of SOX2 -another well-known regulator of self-renewal in cSCCs 14,21 -at the mRNA and protein level, by still undefined mechanisms.It has been shown that SOX2 is required to initiate cSCC tumors in mouse models 21 , and that SOX2 expression is required to sustain tumor growth in mouse and human orthotopic models, by regulating self-renewal 14 .
Surprisingly, when we tested the role of SOX2 inhibition, it did not recapitulate DUSP1 -/+ phenotype or what was observed in cSCCs.Although SOX2 null cells grew slightly slower in 2D conditions, when we grew them in BME, organoids reached greater size and number than controls.Furthermore, when injected into mice, SOX2 KO tumors grew much faster than controls.These data suggest that in the context of tumorigenesis of salivary gland basal cells, SOX2 expression might not be required to promote stemness.In fact, it could be acting as a gatekeeper of premalignant growth, and its loss could promote cancer progression.Additionally, we observed that SOX2 KO tumors lose the expression of transcription factor TP63, which marks basal stem progenitors in the healthy salivary gland 5 .TP63 expression has been described by us to be regulated directly by SOX2 binding to TP63 DNA regulatory regions 16 .We demonstrated that Trp63 silencing promotes apoptosis in cSCC cells and loss of tumor forming potential.This data indicates that A253 cells could become independent of these two TFs during the progression of the disease, and the loss of this TFs promotes the loss of epithelial differentiation.
Overall, these data suggest that DUSP1 acts as an oncogene that drives tumor growth by blocking JNK activity.As for SOX2 expression in these tumors, based on these data we could hypothesize that perhaps it is maintained in earlier stages of the disease, decreasing in later stages what could lead to a decrease in epithelial differentiation.However, how SOX2 expression changes over the course of human disease, remains to be studied in cohorts of patient samples.
Analysis of SOX2 KO RNAseq threw some light into the functions that SOX2 is controlling in these cancers.While DUSP1 KO cells have a decrease in the motility signature, with lower expression of vimentin, fibronectin and ZEB1, SOX2 KO cells have a significant upregulation of these genes (Figure Supp 3F).This is in agreement with what is seen in cSCC, in which Sox2 and Trp63 expression characterize more epithelial tumors, while their downregulation 22 , correlated with the induction of a degree of EMT states, producing tumors with higher stemness and metastatic potential.It is also in agreement with the described function of SOX2 in oral SCC where SOX2 silencing induced a more mesenchymal phenotype 23 .Since EMT TFs have been linked to increased selfrenewal capacity, the SOX2 KO phenotype could be induced by the expression of ZEB1, alone or in combination with other stemness TFs such as KLF4 18 , BMI1 24 or HMGA2, which are also increased in SOX2 KO cells and have been shown to control cancer stem cell stemness.It has been demonstrated that BMI1 functionally marks keratinocytes with cancer stem cell properties in tongue SCCs, and promotes chemotherapy and immunotherapy resistance 25,26 .The induction of EMT has been tightly linked to a metabolic switch 27 .In this regard, we observe that SOX2 KO cells have a decreased OXPHOS signature.Exploring the scRNAseq datasets of mouse SG-SCC -induced by Wnt activation and BMP inhibition-we realized that Dusp1 and Sox2 are upregulated in a specific CSC cell cluster (CSC2) 9 .Importantly, this cluster of cells is also enriched in OXPHOS genes.We hypothesize that these genes are controlled directly by SOX2 expression, since our KO cells are losing the expression of key components of the pathway.Interestingly, we also observed that some of the signature genes enriched in the next population on the trajectory of cancer cells (luminal cells) are upregulated in SOX2 KO cells, suggesting than SOX2 expression would need to decrease to allow the transition.
One of the most striking observations that we made is that SOX2 or DUSP1 loss produce different phenotypes depending on the order of the events.When DUSP1 expression is lost first, although SOX2 expression decreased almost to undetectable levels, the cell death promoted by the activation of JNK seems to be the dominant phenotype.Oppositely, when SOX2 expression is lost first, JNK signaling seems to be impaired regardless of the decrease in DUSP1 expression, its negative regulator, and a pro-tumoral phenotype is predominant.This data perhaps indicates that DUSP1 is required early during carcinogenesis to prevent JNK activation, but once SOX2 expression declines through the progression of the disease, it becomes expendable.How SOX2 RNA decreases upon DUSP1 loss and how JNK signaling is constrained in SOX2 absence remains to be studied.One possibility is that the decrease of SOX2 expression is due to off-target effects of DUSP1 sgRNAs and the constitutive CAS9 used to engineer our cell lines.
In summary, our work demonstrates the role of two relevant genes (DUSP1 and SOX2), in a rare type of cancer such as SG-SCC.DUSP1 acts as an oncogene since its expression is required to constrain JNK signaling and prevent apoptosis.SOX2 on the other hand, has an unexpected role since the loss of its expression enhances both self-renewal and EMT, two well-known processes that drive cancer progression.Overall, these data suggests that SOX2 expression could be a predictor of good prognosis in SG-SCCs.Although these results seem promising, further analyses need to be done in order to explore the expression levels of SOX2 and DUSP1 in patient samples and calculate their correlation with the progression of the disease.
For stable cell line generation, VSV-G pseudotyped lentivirus was produced by PEI transfection of 293 T cells (ATCC) with pLKO sgRNA-carrying vectors and helper plasmids pMD2-VSVg and pPAX2 (Addgene plasmid 12259 and 12260, respectively).293 T cells were maintained in DMEM (Gibco) supplemented with 10% FBS and Pen/Strep solution.Viral supernatant was collected 48 and 72 h after transfection and filtered through 0.45-μm polyvinylidene difluoride filters.For infections, 3 × 10 5 cells were plated in a single well of a 6-well plate, incubated with a 1:2 dilution of viral supernatant containing 8 μg/mL Polybrene.Forty-eight hours after infection, puromycin-resistant cells were selected with 1 μg/mL puromycin (Sigma-Aldrich).
Growth curves of cultured cells were measured by crystal violet staining and reported as p < 0.05 calculated by Student's t test.Cells were exposed to 20 J/m 2 of U.V.C and protein collected at the indicated time points after exposure.A253 control and DUSP1KO or sgSOX2 cells (1 × 10 5 cells/injection) were prepared in 50% Matrigel and injected intradermally in Nude recipient mice.

CRISPR/Cas9 knockouts in SCC cells
The DUSP1 exon2 was deleted with two sgRNAs targeting the 5´and 3´ intronic regions of the gene.SOX2 was targeted with two independent sgRNAs guided to the begging of SOX2 exon.The most efficient guide RNAs were predicted using the CRISPR design tool from Benchling.sgRNA against dTomato was used as a control.sgRNA sequences were cloned into pLKO U6-puro vector (Addgene 52963).After sgRNA transduction and selection, cells were subsequently infected with lentiCas9-Blast (Addgene 52962) and selected with Blasticidin for 3 days (5 μg/ml).Single cell clones were selected and screened by PCR of genomic DNA.Clones were further validated by Western blot analysis with anti-DUSP1 antibodies.sgRNA sequences are listed in Table Supp.3.

Scratch assay
500,000 cells were seeded in 6-well plates.Cells were treated with 8 ug/ml of mitomycin for 2 h.Subsequently, wounds were created using a 200ul pipette tip, washed with 1 × PBS, and incubated in fresh medium.Images were taken at 0, 16, 24 and 48 h until scratch closed.ImageJ was used to quantify the area of each scratch, and the percentage of closure was normalized to time 0.

Apoptosis and cell cycle assays on organoids
Organoids were retrieved by dissolving BME with Dispase II (Roche) (100 mg/ml stock) diluted 1:100 in ADV + + + medium.Rock inhibitor was also added to a final concentration of 10 uM.Organoids were incubated at 37 °C for 30 min and retrieved in a 15 ml tube with equal volume ADV+++ and a p1000 tip cut at the end.ADV medium was added until 10 ml, and tubes containing the organoids were incubated on ice for at least 20 min.Organoids were centrifuged at 300 g 5 min at 4 °C, supernatant was removed, and pellet was once again resuspended with up to 10 ml ADV medium, and left on ice another additional 20 min.After centrifugation, 1 mL TrypleExpress was added cells incubated at 37 °C for 5 min to digest organoids into single cell suspension.10% FBS was added to stop digestion and centrifuged.Supernatant was removed and pellet was washed with 1X

Quantitative reverse-transcription PCR
mRNA was isolated using Qiazol (Qiagen) and Direct-zol RNA Mini Prep Kits (R2052, Zymo Research).Samples were quantified using a Nanodrop spectrophotometer (Thermo Scientific).Complementary DNA was synthesized from 1.5 μg of total RNA using NZY First-Strand cDNA Synthesis Kit with random primers (NZYTech).qRT-PCR was performed with KAPA SYBR® FAST (Rox) (KK4602, Kapa) on a StepOnePlus™ Real-Time PCR System (Applied Biosystems).Measurements were recorded in triplicate.Differences between samples and controls were calculated based on the 2 −ΔΔCT method and normalized to RPLP0.For detailed list of primer sequences, see Table Supp

RNA-seq library preparation
Total RNA was extracted from 2 × 10 5 control or CRISPR engineered cells using the RNA Microprep Kit (Zymo) according to manufacturer's instructions.RNA quality was defined using an Agilent 2100 Bioanalyzer before we prepared Poly(A) + selected, multiplexed, paired end libraries with the Illumina TruSeq RNA preparation kit.

Figure 5 .
Figure 5. SOX2 KO SG-SCC suffer an epithelial to mesenchymal transition.(A) Heatmap illustration the DEG between sgTomato and sgSOX2 A253 cells.(B) Graph showing the gene ontology biological processes (left) or pathways (right) enriched or depleted in sgSOX2.2cells.In bold are highlighted the most relevant findings.(C) Bar graphs showing the decreased expression (Normalized counts) of squamous differentiation genes in sgSOX2.2cells.(D) Left panel, Confocal micrographs illustrating KRT10 positivity (red) within a6 integrin positive (white) tumor cells.Right panel, Violin plots quantifying the intensity (right panel) or area (right panel) of KRT10 on sgTomato and sgSOX2 tumors (One-way ANOVA).(E) Bar graphs showing the increased expression (Normalized counts) of mesenchymal genes in sgSOX2.2cells.(F) Micrographs showing vimentin staining in sgTomato and sgSOX2 organoids.(G) Box plot illustrating the % of wound closure in sgTomato and sgSOX2 cells (n = 3, t-test).(H) Left, Confocal micrographs demonstrating a decreased TP63 expression (red) in sgSOX2.2grown tumors in comparison with sgTomato controls.a6-integrin (white) marks cancer cells; right, Violin plots representing the measurement of TP63 nuclear intensity within the a6-integrin positive cancer cells (one-way ANOVA).I. Bar graphs showing the increased expression (Normalized counts) of self-renewal transcription factors in sgSOX2.2cells.Scale bars, 50 µm.

Mice 6 -
week-old, female Nude (NU/NU [088] Charles River) mice were used for orthotopic transplantations and xenograft studies.Tumors were detected by palpation, measured with digital calipers to calculate tumor volumes (V Tumor = (π/6) x l x w2 , where l = length in mm and w = width in mm).All animal experiments were performed in accordance with the guidelines and approval of the Institutional Animal Care and Use Committee at Instituto Investigaciones Biomédicas Alberto Sols.All authors complied with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines.All mice were grown under circadian light (12 h/12 h) at (22 ± 2) °C with freely available clean water and feed. 3.